Browsing Theses and Dissertations School of Pharmacy by Title "Mechanism of action of 1-beta-D arabinofuranosyl cytosine (Ara-C): The biological consequences of the incorporation of Ara-C into a eukaryotic DNA replication origin"
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Mechanism of action of 1-beta-D arabinofuranosyl cytosine (Ara-C): The biological consequences of the incorporation of Ara-C into a eukaryotic DNA replication origin1-beta-D arabinofuranosyl cytosine (Ara-C) is a highly effective drug used to treat acute leukemia. Yet, despite its high efficacy, the precise mechanism of action of ara-C mediating the cell killing and inhibition of DNA synthesis remains unclear. Ara-C is converted to the active form, ara-CTP, by cellular kinases. Ara-CTP inhibits the DNA synthesis by inhibiting DNA polymerase and blocks the elongation of the DNA strand. However, the studies using intact cells and viral system indicate that ara-C is mainly incorporated into DNA at internucleotide linkages and ara-C slows but does not terminate the DNA synthesis, suggesting the alternative mechanism of action of ara-C. We have investigated the biological effects of the incorporation of ara-Cinto DNA using the SV40 DNA replication system. Ara-C incorporation into SV40 origin caused a site specific inhibition of T-Ag (an initiator protein of DNA replication) and RP-A (human single stranded DNA binding protein) binding to the origin DNA, and reduction in the stability of DNA. Ara-C substitution at T-Ag binding site I domain inhibited more effectively the binding of T-Ag and promoted greater reduction in the stability of DNA duplexes compared to the ara-C substitution at T-Ag binding site II and early mRNA domains, and contributes to the ara-C substitutions at other sites in inhibiting T-Ag binding and reducing the stability of the DNA duplex. The ara-C substitutions at both site I and early mRNA domains also disrupted RP-A binding. Furthermore ara-C substituted origin DNA has lower affinity for T-Ag binding than unsubstituted DNA. These site specific effects of ara-C incorporation may be sequence dependent. Moreover, ara-C incorporation within the SV40 origin appears to have essentially no effect on the enzymatic unwinding of origin DNA by T-Ag helicase and the initiation and subsequent elongation of the origin DNA by the human multiprotein DNA replication complex (MRC). The data suggest that the ara-C incorporation changes the structure of DNA, which alters the binding affinity of the origin DNA for T-Ag and RP-A binding, but does not disrupt the enzymatic processes involved in replication of the DNA. Our results support the findings that: (1) ara-C is a weak competitive inhibitor of DNA polymerase; and, (2) ara-C incorporation slows, but does not terminate DNA synthesis.